All organisms respond to extreme environmental conditions by either inducing de novo or dramatically increasing the expression of a number of genes that protect the cell from the deleterious effect of intracellular protein denaturation. These genes encode for a family of proteins called HSPs (heat shock proteins) and other molecular chaperones and cytoprotective proteins. Expression of HSPs and other chaperones is induced upon exposure to a variety of stressors including elevated temperature, oxidative stress, alcohol, hyper- and hypoosmotic stress, transition metals, viral infection, amino acid analogs, etc. (Morimoto, et al., In The Biology of Heat Shock Proteins and Molecular Chaperones, 1994 (New York: Cold Spring Harbor Press), pp. 417-455). HSPs are involved in basic cellular processes under both stress and normal conditions such as correct folding of nascent polypeptides, binding to exposed hydrophobic regions of denatured or abnormal proteins to prevent their aggregation and promote degradation, and translocation of proteins into membrane-bound organelles in the cell. Expression of some HSPs is essential during embryogenesis (Luft, et al., Chaperones. 1999; 4:162-170). In addition, certain HSPs are required to stabilize otherwise unstable proteins or complexes of proteins. The signal-transducing proteins such as steroid hormone receptors (Pratt, Annu. Rev. Pharmacol. Toxicol. 1997; 37:297-326) and protein kinases (Donze, et al., Mol. Cell Biol. 1999; 19:8422-8432; Louvion, et al., Mol. Biol. Cell 1998; 9:3071-3083; Zhu, et al., Development 1997; 124:3007-3014) have been the most extensively investigated such proteins.
HSP family proteins are classified into several groups based on their molecular weight and sequence homology. In mammals, major HSP classes are HSP70, HSP40, HSP90, and HSP27. HSP70 subfamily includes molecular chaperones that participate in folding of nascent polypeptide chains (Mayer, et al., Adv. Protein Chem. 2001; 59:1-44), unfolding and refolding of proteins during their transport across membranes (Jensen, et al., Curr. Biol. 1999; 9:R779-R782; Pilon, et al., Cell 1999; 97:679-682; Ryan, et al., Adv. Protein Chem. 2001:59:223-242), binding to partially denatured, abnormal, or targeted for proteasome degradation proteins (Zylicz, et al., IUBMB. Life 2001; 51:283-287). Bacterial counterpart for HSP70 is DnaK protein. HSP70 subfamily includes both constitutive and stress-inducible proteins that are closely related and often referred to as Hsc70 and HSP72 respectively. HSP40 is a co-chaperon for HSP70 class proteins, which modulates ATPase activity and substrate binding properties of the latter. Its bacterial analog is DnaJ (Ohtsuka, et al., Int. J. Hyperthermia 2000; 16:231-245). HSP90 is ubiquitously expressed and may constitute up to 1-2% of total cellular protein. Mammalian cells express at least two HSP90 isoforms, HSP90α and HSP90β, which are encoded by two separate genes (Pearl, et al., Adv. Protein Chem. 2001; 59:157-186). Under stress conditions HSP90 binds to exposed hydrophobic regions of denatured proteins, while in the absence of stress it participates in fundamental cellular processes such as hormone signaling and cell cycle control (Pearl, et al., Curr. Opin. Struct. Biol. 2000; 10:46-51). Many regulatory proteins, including steroid hormone receptor, cell cycle kinases, and p53 have been identified as HSP90 substrates (Young, et al., J. Cell Biol. 2001; 154:267-273; Pratt, Annu. Rev. Pharmacol. Toxicol. 1997; 37:297-326). HSP25/27 belongs to a family of small heat shock proteins, which includes primate HSP27, rodent HSP25, αA-crystallins and αB-crystallins. HSP25/27 is expressed constitutively and expression increases after exposure to heat, transition metal salts, drugs, and oxidants. It forms oligomers consisting of 8-40 monomers that serve as binding sites for unfolding peptides until they can be refolded by HSP70/HSP40 system (Van Montfort, et al., Adv. Protein Chem. 2001; 59:105-156; Welsh, et al., Ann. N.Y. Acad. Sci. 1998; 851:28-35).
The induction of HSP gene expression occurs primarily at transcriptional level and is mediated by a family of transcription factors named HSF (Heat Shock Factor). Whereas vertebrates and plants have at least four members of the HSF family, only one HSF is expressed in yeast, Drosophila, and C. elegans (Wu, Ann. Rev. Cell Dev. Biol. 1995; 11:441-469). In human cells, three HSFs (HSF1, HSF2, and HSF4) have been characterized (Morimoto, et al., Genes Dev. 1998; 12:3788-3796). HSF1 is ubiquitously expressed and plays the principle role in the stress-induced expression of HSPs. It is an apparent functional analog of Drosophila HSF.
Under normal conditions, HSF1 exists in the cell as an inactive monomer. Following exposure to elevated temperature, HSF1 trimerizes and apparently relocates to the nucleus where it binds to specific sites in HSP promoters upstream of the transcription initiation site (Wu, Ann. Rev. Cell Dev. Biol. 1995; 11:441-469; Westwood, et al., Mol. Cell Biol. 1993; 13:3481-3486; Westwood, et al., Nature 1991; 353:822-827). The HSF binding site contains arrays of inverted repeats of the element NGAAN designated HSE (Heat Shock Element). The same evolutionarily conserved HSE sequence is recognized by all members of the HSF protein family and is universal to all eukaryotic species (Kim et al., Protein Sci. 1994; 3:1040-1051). The heat shock promoter is primed for rapid activation in response to heat shock. Many factors of the basal transcription machinery are bound to the promoter prior to heat shock including GAGA factor, TFIID, transcriptionally arrested RNA polymerase II located 21-35 nucleotides downstream of the transcription start site, and presumably some other transcription factors (Shopland, et al., Genes Dev. 1995; 9:2756-2769). The partitioning of HSF molecules between the nucleus and cytoplasm is a subject to some controversy since, in the case of Xenopus laevis, HSF1 was shown to be a nuclear protein before heat shock (Mercier, et al., J. Biol. Chem. 1997; 272:14147-14151), while in most studies employing mammalian cells, HSF1 was found in both the cytoplasm and nucleus under normal conditions (Sarge, et al., Mol. Cell Biol. 1993b; 13:1392-1407). Interestingly, heat shock treatment of HeLa cells results in rapid and reversible localization of HSF1 in specific nuclear granules, which constitute a novel type of nuclear protein compartmentalization (Cotto, et al., Journal of Cell Science 1997b; 110:2925-2934). The granules appear within 30 sec of heat shock treatment and rapidly disappear upon shift to normal temperature. However, the functional significance of this phenomenon is still unknown.
The overall structure of HSF1 is conserved among species as distant as Drosophila and human. The DNA-binding domain is just over 100 amino acids long and is situated close to the N-terminus of the molecule. This domain is about 70% homologous between human HSF1 and Drosophila HSF and shows 55% homology between human HSF1 and yeast HSF. The leucine zipper domain, which is C-terminal with respect to the DNA-binding domain, is even more conserved showing 79% homology between human and Drosophila. In vertebrates, this domain comprises three hydrophobic heptad repeats with an additional heptad repeat located in the C-terminus of HSF1. It has been implicated in the maintenance of the inactive monomeric state of HSF1 under non-stressful conditions (Wu, et al., In The Biology of Heat Shock Proteins and Molecular Chaperones, 1994 (New York: Cold Spring Harbor Press), pp. 395-416).
A number of models have been proposed to explain how HSF activation is regulated, most of them focusing on repression of the inactive monomer under normal conditions as the most probable mode of regulation. Several lines of evidence suggest the existence of a titratable cellular factor that acts to repress HSF under normal conditions by keeping it in a monomeric form. Indeed, overexpression of both HSF1 and HSF2 in 3T3 mouse fibroblasts resulted in constitutive activation of their DNA-binding activity and transcription of HSP genes (Sarge, et al., Mol. Cell Biol. 1993a; 13:1392-1407). The observed effect could reflect either general cellular stress caused by the drastic increase in HSF concentration or titration of the negative regulator of HSF, which is present in limiting amounts. Furthermore, expression of human HSF1 in Drosophila cells results in a decrease of the activation threshold temperature by 9 degrees, to the temperature characteristic for the heat shock conditions in Drosophila (32° C.) instead of 41° C.—a characteristic threshold for mammalian cells. At the same time, Drosophila HSF expressed in human cells remained constitutively active even when the temperature was lowered to 25° C.—the normal growth temperature for Drosophila (Clos, et al., Nature 1993; 364:252-255). Similarly, Arabidopsis HSF remained active in Drosophila and human cells even under control conditions (Hubel, et al., Mol. Gen. Genet. 1995; 248:136-141). Taken together, these results strongly suggest that the intracellular environment rather than structure of the HSF molecule determines its behavior in response to heat stress. These data are consistent with the possibility that HSF activation is mediated by a specific stimulating factor(s).
The process of HSF1 activation can be divided into at least two steps: 1) trimerization and acquisition of DNA binding activity; 2) acquisition of transactivation competence, which is correlated with hyperphosphorylation of the factor. Treatment with salicylate and other non-steroid anti-inflammatory drugs induces HSF trimerization and DNA binding but fails to stimulate transcription of HSP genes (Jurivich, et al., J. Biol. Chem. 1995a; 270:24489-24495). However, majority of HSF regulation occurs at the level of its trimerization.
The model of HSF regulation, where HSF activity is a subject to the negative feedback mechanism involving inducible HSP72 and other chaperones, has been a paradigm for a decade. According to this model, HSF monomer is present in the complex with HSP72 and other chaperones (most notably HSP90) under normal conditions. Trimerization of HSF molecules is thought to occur spontaneously as soon as negative regulation by HSPs has been relieved. Indeed, in a number of studies HSF has been shown to possess intrinsic responsiveness to heat (Zuo, et al., Mol. Cell Biol. 1995; 15:4319-4330; Farkas, et al., Molecular and Cellular Biology 1998a; 18:906-918). However, the HSF concentrations used in these studies far exceeded those found in the cell, which questions the physiological relevance of the data. Furthermore, although all these studies imply that HSF trimerization occurs spontaneously once the negative regulation is relieved, the existence of a positive regulation of HSF activity can not be ruled out. For example, the rapid, specific and reversible formation of HSF granules in nuclei during heat shock (Cotto, et al., Journal of Cell Science 1997a; 110:2925-2934; Jolly, et al., Journal of Cell Science 1997; 110:2935-2941) testifies against spontaneous mechanism given the relatively low number of HSF molecules in the cell, their even distribution throughout cytoplasm under normal conditions, and molecular crowding effect due to very high total protein concentration in the cell as compared to in vitro experimental systems.
HSPs, and HSP70 family in particular, is considered a part of a protective mechanism against certain pathological conditions, including ischemic damage, infection, and inflammation (Pockley, Circulation 2002; 105:1012-1017). In the case of inflammation, a protective role of HSPs has been shown in a variety of experimental models (Jattela et al., EMBO J. 1992; 11:3507-3512; Morris et al., Int. Biochem. Cell Biol. 1995; 27:109-122; Ianaro et al., FEBS Lett. 2001; 499:239-244; Van Molle et al., Immunity 2002; 16:685-695). For example, Ianaro et al. (Mol. Pharmacol. 2003; 64:85-93) have recently demonstrated that HSF1-induced (see below) HSP72 expression in the inflamed tissues and activation of the heat shock response is closely associated with the remission of the inflammatory reaction. It follows, that HSP genes may function as anti-inflammatory or “therapeutic” genes, and their selective in vivo transactivation may lead to remission of the inflammatory reaction (Ianaro et al., FEBS Lett. 2001; 499:239-244 and Ianaro et al., FEBS Lett. 2001; 508:61-66).
A potential therapeutic value of causing increased expression of HSPs in individuals suffering from cerebral or cardiac ischemia and neurodegenerative diseases has been also suggested (Klettner, Drug News Perspect. 2004; 17:299-306; Hargitai et al., Biochem. Biophys. Res. Commun. 2003; 307:689-695; Yenari et al., Ann. Neurol. 1998; 44:584-591; Suzuki et al., J. Mol. Cell Cardiol. 1998; 6:1129-1136; Warrik et al., Nat. Genet. 1999; 23:425-428). For example, Zou et al. (Circulation 2003; 108:3024-3030) have recently shown that cardiomyocyte cell death induced by hydrogen peroxide was prevented by overexpression of HSF1 in COS7 cells. Thermal preconditioning at 42° C. for 60 minutes activated HSF1, which played a critical role in survival of cardiomyocytes from oxidative stress. Ischemia/reperfusion injury has been reported to induce apoptosis in cardiomyocytes (Fliss and Gattinger, Circulation 1996; 79:949-956). Zou et al. (Circulation 2003; 108:3024-3030) have also demonstrated that, in the heart of transgenic mice overexpressing a constitutively active form of HSF1 (and having elevated levels of HSPs 27, 70 and 90 in the heart), ischemia followed by reperfusion-induced ST-segment elevation in ECG was recovered faster, infarct size was smaller, and cardiomyocyte death was less than in wild-type mice. These results suggest that elevated activity of HSF1 (and levels of HSPs) exert protective effect on the electrical activity of myocardium against ischemia/reperfusion injury (see also Plumier et al., J. Clin. Invest. 1995; 95:1854-1860; Marber et al., ibid., pp. 1446-1456; Radford et al., Proc. Natl. Acad. Sci. USA, 1996; 93:2339-2342).
HSPs and HSF1 have been also implicated in protection against a variety of neurodegenerative disorders that involve aberrant protein folding and protein damage. Neuronal cells are particularly vulnerable in this sense as HSF activity and HSP expression are relatively weak in such cells and motor neurons appear to require input of HSP secreted from adjacent glial cells to maintain adequate molecular chaperone levels (Batulan et al., J. Neuosci. 2003; 23:5789-5798; Guzhova et al., Brain Res. 2001; 914:66-73).
Polyglutamine (polyQ) expansion is a major cause of inherited neurodegenerative diseases called polyglutamine diseases. Several polyQ diseases have been identified, including Huntington's disease (HD), spinobulbar muscular atrophy, dentatorubral pallidoluysian atrophy, Kennedy disease, and five forms of spinocerebellar ataxia. Aggregates or inclusion bodies of polyQ proteins (e.g., huntingtin) within the nucleus, or in the cytoplasm of neuronal cells in some Huntington's disease patients, are a prominent pathological hallmark of most polyQ diseases (Davies et al., Cell 1997; 90:537-548; DiFiglia et al., Science 1997; 277:1990-1993). Formation of polyQ protein inclusions correlates with an increased susceptibility to cell death (Warrik et al., Cell 1998; 94:939-49). The early stages of aggregates are highly toxic to cells (Bucciantini et al., Nature 2002; 416:507-11). Suppression of aggregates should be beneficial to cells and could delay the progression of polyQ diseases (Sanchez et al., Nature 2003; 421:373-9). HSPs have been implicated in many of these neurodegenerative diseases based on the association of chaperones with intracellular aggregates. For example, live cell imaging experiments show that Hsp70 associates transiently with huntingtin aggregates, with association-dissociation properties identical to chaperone interactions with unfolded polypeptides (Kim et al., Nat. Cell Biol. 2002; 4:826-31). This suggests that these chaperone interactions may reflect the efforts of Hsp70 to direct the unfolding and dissociation of substrates from the aggregate. Moreover, overexpression of the Hsp70 chaperone network suppresses aggregate formation and/or cellular toxicity. A critical protective role for small HSPs (HSP27) has been also recently demonstrated in Huntington's disease (Wyttenbach et al., Human Mol. Gen. 2002; 11:1137-51). Collectively, these observations have led to the hypothesis that the elevated levels of heat shock proteins reduce or dampen aggregate formation and cellular degeneration (Warrick et al., Nat. Genet. 1999; 23:425-8; Krobitsch and Lindquist, Proc. Natl. Acad. Sci. USA 2000; 97:1589-94). Importantly, HSF1 overexpression suppressed polyQ-inclusion formation even better than any combination of HSPs in culture cells and in Huntington's disease model mice extending their life span (Fujimoto et al., J. Biol. Chem. 2005; 280:34908-16).
Multiple HSPs are also overexpressed in brains from Alzheimer's (AD) and Parkinson's disease (PD) patients and found to be associated with senile plaques and Lewy bodies, respectively. These HSPs may be involved in neuroprotection by various mechanisms ranging from microglia activation and clearance of amyloid-β peptides to suppression of apoptosis (Kitamura and Nomura, Pharmacol Ther. 2003; 97:35-33).
Ageing is also associated with the decrease in activity of HSF (Tonkis and Calderlwood, Int. J. Hyperthermia 2005; 21:433-444). Indeed, neurodegenerative diseases often occur later in life when heat shock genes seem to be induced poorly (Soti and Csermely, Exp. Gerontol. 2003; 38:1037-40; Shamovsky and Gershon, Mech. Ageing Dev. 2004; 125:767-75). Moreover, it has been recently shown that induction of heat shock either by temperature or HSF overexpression could extend life span in model organisms. For example, the heat shock response has recently been implicated in the regulation of longevity in Caenorhabditis elegans in a pathway that overlaps with the insulin signaling pathway (Hsu et al., Science 2003; 300:1142-5; Morley and Morimoto, Mol. Biol. Cell 2004; 15:657-64). Reduction of HSF1 levels cause a decreased life span in C. elegans, similar to life span effects observed in mutants of Daf-16, a FOXO transcription factor in the insulin signaling pathway. Daf-16 and HSF1 share a subset of downstream target genes, including small HSPs. RNA interference experiments showed that a decrease in small HSPs and other HSPs leads to a decrease in longevity (Hsu et al., Science 2003; 300:1142-5; Morley and Morimoto, Mol. Biol. Cell 2004; 15:657-64). Similarly, Walker et al. (Aging Cell 2003; 2:131) have demonstrated that overexpression of HSP16 can extend C. elegans' life span. Therefore, in addition to the prevention of diseases of aging, increased levels of HSPs may lead to increases in life span (Westerheide and Morimoto, J. Biol. Chem. 2005, 280:33097-100).
Heat shock is a known transcriptional activator of human immunodeficiency virus type 1 (HIV) long terminal repeat (LTR). However, HIV LTR suppression can occur under hyperthermic conditions. Specifically, suppression of the HIV LTR was observed in a conditional expression system for gene therapy applications that utilizes the heat-inducible promoter of the human heat shock protein 70B gene to enhance the HIV LTR-driven therapeutic gene expression after hyperthermia (temperature higher than 37° C.) (Gemer et al., Int. J. Hyperthermia 2000; 16:171-181). Similarly, the inhibition of HIV transcription has been reported after whole-body hyperthermia at 42° C. in persons with AIDS (Steinhart et al., J. AIDS Hum. Retrovirol. 1996; 11:271-281). Recently demonstrated ability of a mutant transcriptionally active HSF1 (lacking C-terminal residues 203-315) to suppress HIV promoter activity further suggests that HSF1 could serve as a tool for the treatment of AIDS (Ignatenko and Gemer, Exp. Cell Res. 2003; 288:1-8; see also Brenner and Wainberg, Expert Opin. Biol. Ther. 2001; 1:67-77).
Due to interaction of HSPs with numerous regulatory proteins (e.g., NF-κB, p53, v-Src, Raf1, Akt, steroid hormone receptors) and pathways (e.g., inhibition of c-Jun NH2-terminal kinase (JNK) activation, prevention of cytochrome c release, regulation of the apoptosome, prevention of lysosomal membrane permeabilization, prevention of caspase activation) involved in the control of cell growth, the increased production of HSPs has potent anti-apoptotic effect (Bold, et al., Surgical Oncology-Oxford 1997; 6:133-142; Jaattela, et al., Exp. Cell Res. 1999; 248:30-43; Nylandsted, et al., Ann. N.Y. Acad. Sci. 2000; 926:122-125; Pratt and Toft, Exp. Biol. Med. (Maywood) 2003; 228:111-33; Mosser and Morimoto, Oncogene 2004; 23:2907-18). Anti-apoptotic and cytoprotective activities of HSPs directly implicate them in oncogenesis (Jolly and Morimoto, J. Natl. Cancer Inst. 2000; 92:1564-72; Westerheide and Morimoto, J. Biol. Chem. 2005, 280:33097-100). Many cancer cells display deregulated expression of HSPs, whose elevated levels contribute to the resistance of cancerous cells to chemo- and radiotherapy. Different subfamilies of HSPs including HSP70, HSP90, and HSP27 were found to be expressed at abnormal levels in various human tumors (Cardoso, et al., Ann. Oncol. 2001; 12:615-620; Kiang, et al., Mol. Cell Biochem. 2000; 204:169-178). In some cases, HSPs are expressed at cell surface in tumors, most probably serving as antigen presenting molecules in this case (Conroy, et al., Eur. J. Cancer 1998; 34:942-943). Both HSP70 and HSP90 were demonstrated to mediate cytoplasmic sequestration of p53 in cancer cells (Elledge, et al., Cancer Res. 1994; 54:3752-3757). Inactivation of wild-type p53 function has been observed in variety of cancer cells and is in fact one of the most common hallmarks in human cancer (Malkin, et al., J. Neurooncol. 2001; 51:231-243). Other studies have demonstrated that HSP70 has a potent general antiapoptotic effect protecting cells from heat shock, tumor necrosis factor, serum starvation, oxidative stress, chemotherapeutic agents (e.g., gemcitabine, torootecan, actinomycin-D, campothecin, and etoposide), and radiation (Jaattela, et al., EMBO J. 1992; 11:3507-3512; Jaattela, et al., J. Exp. Med. 1993; 177:231-236; Simon, et al., J. Clin. Invest 1995; 95:926-933; Karlseder, et al., Biochem. Biophys. Res. Commun. 1996; 220:153-159; Samali and Cotter, Exp. Cell Res. 1996; 223:163-170; Sliutz et al., Br. J. Cancer 1996; 74:172-177). At the same time, HSP70 is abundantly expressed in human malignant tumors of various origins, not only enhancing spontaneous growth of tumors, but also rendering them resistant to host defense mechanisms and therapeutic treatment (Ciocca, et al., Cancer Res. 1992; 52:3648-3654). Therefore, finding a way to suppress HSP overproduction in cancerous cells will be invaluable for increasing the efficacy of the existing anti-cancer therapeutic approaches.
HSF1-mediated induction of HSPs has been also implicated in protection of sensory hair cells against acoustic overexposure, hyperthermia and ototoxic drugs. It has been shown that mice lacking HSF1 have reduced recovery from noise-induced hearing loss (Altschuler et al., Audiol Neotol. 2003; 7:152-156). Similarly, Sugahara et al. (Hear Res. 2003; 182:88-96) have demonstrated that the loss of sensory hair cells was more significant in HSF1-null mice compared with that of wild-type mice when mice were subjected to acoustic overexposure. They have also shown that the loss of both the sensory hair cells and the auditory function induced by acoustic overexposure was inhibited by pretreatment of the inner ear with local heat shock.
Based on the information provided above, HSF1 appears to be an attractive therapeutic target for regulating HSP synthesis to combat cancer, inflammation, ischemia, neurodegeneration, age-related diseases, HIV infection, deafness, and related disorders (Mestril et al., J. Clin. Invest. 1994; 93:759-67; Morimoto, et al., Genes Dev. 1998; 12:3788-3796; Jolly and Morimoto, J. Natl. Cancer Inst. 2000; 92:1564-72; Ianaro et al., FEBS Lett. 2001; 499:239-44; Calderwood and Asea, Int. J. Hyperthermia 2002; 18:597-608; Zou et al., Circulation 2003; 108:3024-30; Westerheide and Morimoto, J. Biol. Chem. 2005; 280:33097-100).